11.02 Just an Appendectomy? Outcomes of Appendectomy in Patients with End-Stage Renal Disease

Posted on December 22, 2014

M. C. Smith1, M. R. Boylan2,3, R. Lee1, A. E. Alfonso1, G. Sugiyama1  1SUNY Downstate Medical Center,Department Of Surgery,Brooklyn, NY, USA 2SUNY Downstate School Of Public Health,Department Of Epidemiology And Biostatistics,Brooklyn, NY, USA 3SUNY Downstate College Of Medicine,Brooklyn, NY, USA

Introduction:  With each passing year, the number of patients with end-stage renal disease (ESRD) is steadily increasing. Due to improvements in management, these patients are undergoing common general surgical procedures more frequently. There is limited data on the association between ESRD and outcomes following common general surgical procedures such as appendectomy.

Methods:  We selected patients from the Nationwide Inpatient Sample between 1998 and 2010 who underwent appendectomy. We used ICD-9 codes to identify patients with ESRD and to track perioperative complications during admission. Data on patient demographics, length of stay, total hospital charges, and mortality during admission were also extracted. Our statistical models were logistic and linear regressions that controlled for age, gender, race, insurance type, and number of Elixhauser comorbidities.

Results: Our study population included 7,519 patients with ESRD, with the remaining 3,638,041 patients serving as controls. In patients with ESRD, 56% of cases of appendicitis involved perforation, compared to 28% in controls (p<0.001). Laparoscopic appendectomy was performed in only 34% of ESRD patients, compared to 47% of controls (p<0.001), with the remaining patients receiving open appendectomy. Patients with ESRD were at a 72% increased overall risk of complication (OR=1.72; 95% CI, 1.51-1.96; p<0.001), and were particularly at increased risk of death (OR=10.97; p<0.001), mechanical wound complication (OR=2.46; p<0.001), and infection (OR=1.51; p=0.008). Mean length of stay was 12.42 days in patients with ESRD, compared to 3.09 days in controls (p<0.001). Mean total charges were $91,876 in patients with ESRD, compared to $21,409 in controls (p<0.001).

Conclusion: In patients with acute appendicitis requiring appendectomy, ESRD was observed to be an independent risk factor for adverse short-term postoperative outcomes including death, infection, increased length of stay, and increased hospital charges. It is important for surgeons to be vigilant in their care of these patients in order to anticipate and mitigate the effects of these complications.

 

11.03 A Systems Engineering Evaluation to Readmission Following Complex Surgery

Posted on December 22, 2014

A. W. Acher1,4, T. J. LeCaire1,4, A. S. Hundt2, C. C. Greenberg1,2,3,4, P. Carayon2,3, S. M. Weber1,4  4University Of Wisconsin,School Of Medicine And Public Health,Madison, WI, USA 1University Of Wisconsin,Wisconsin Surgical Outcomes Research Program, Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Center For Quality & Productivity Improvement,Madison, WI, USA 3University Of Wisconsin,Department Of Industrial And Systems Engineering, College Of Engineering,Madison, WI, USA

Introduction: Prior studies on readmission neglect the patient perspective. This study sought to fill this gap by applying the Systems Engineering Initiative for Patient Safety (SEIPS) model, which places the patient at the center of the system.  The objective was to identify system factors that contribute to or mitigate the risk of readmission.

Methods: Patients readmitted within 30 days of discharge (n=12) participated in a semi-structured interview with questions guided by the SEIPS framework on post-operative care, preparation for discharge, and etiology of readmission. Audio-recorded interviews were transcribed. Content analysis using a SEIPS-based coding framework was performed. Pre-specified data elements were collected concurrently from the medical record for a mixed-methods analysis.

Results: Index operations included colorectal, pancreatectomy, esophagectomy, and liver resection. Patients were readmitted a mean of 9.8 days post-discharge (SD: +/-7.3, median 8.5). Readmission diagnoses included: infection, small bowel obstruction, dehydration, pleural effusion, possible hemorrhage and palliative care. Patients identified a number of factors during transition of care from inpatient to home that may have contributed to readmission. Common findings centered on care team structure and communication, and educational process and content (Table).

Only 1/3 of patients felt they remembered all or most of their discharge instructions. Patients felt “pressured to hurry up and get it done” and a “rush to get you out of here” with insufficient time for questions. Information provided was too expansive (“so much paperwork”) with not enough “clear instruction” about specific clinical issues. Many patients (n=5) reported only being somewhat confident to prevent problems at home.

While 42% felt their care team communicated well, the remaining said “I think so” (n=4), were unsure (n=1) or disagreed (n=2). Patients (n=5) reported care team members were “not on the same page” with “every person [having] a different story about what they were going to do.” Several patients would have preferred more communication from their surgeon rather than discordant information from multiple sources.

Conclusion: This is the first study to utilize a systems engineering approach to assess the etiology of readmission from the patient perspective. Following complex surgery, readmitted patients note issues in care team structure and communication, as well as educational process and content, that may contribute to readmission. Future interventions should focus on these areas to enhance the transition of care and potentially decrease readmissions.

11.04 Predictors of Mortality in Abdominal Compartment Syndrome from the National Inpatient Sample

Posted on December 22, 2014

S. Ross1, P. Fischer1, K. Dahey1, C. Huntington1, T. Cox1, L. Blair1, B. T. Heniford1, V. Augenstein1, A. B. Christmas1, R. Sing1  1Carolinas Medical Center,Charlotte, NC, USA

Introduction:
Abdominal compartment syndrome (ACS) can lead to multisystem organ dysfunction, often requiring surgical decompression despite early medical management. To date, no national, multicenter data exists regarding patient treatment, outcomes, and risk factors for mortality in ACS. Our purpose was to quantify rates of adverse outcomes in ACS and identify predictors of mortality.

Methods:
The National Inpatient Sample database was queried from 2006-2011 for all traumatic and non-traumatic ICD-9 diagnoses of ACS. Primary outcomes were medical and procedural complications and in-hospital mortality. Standard statistical tests were applied, and stepwise logistic regression (SLR) was used to identify independent predictors of mortality.

Results:
There were 2,657 patients with ACS: 2,077 from non-traumatic and 580 from traumatic etiologies. On average, patients were middle aged (56.4 ± 17.7 years), male (63.0%), had few comorbidities (Charlson Comorbidity Index 1.8 ± 2.2). Concomitant abdominal operations were performed in 63.4% of patients including exploration and/or resection. Causes of complications were medical in 95.1% and surgical in 52.1%, with overall mortality rates high at 50.7%. Patients with traumatic ACS had lower rates of medical complications (77.8%vs100%;p<0.001), but similar surgical complication (53.1%vs53.8%; p>0.05) and mortality rates (48.8%vs51.5%; p>0.05). Significant univariate analysis results by mortality are reported in the table.  On SLR, predictors of mortality (odds ratio): emergent admission (1.5), teaching hospital (1.8), increasing age (1.03/year).  Protective factors by SLR were: elective admission (0.66), surviving to tracheostomy (0.32), gastrostomy (0.36), and ventral hernia repair (0.13); p<0.001 for all.

Conclusion:
Abdominal compartment syndrome carries significant morbidity with over half of cases resulting in death.  Patients who survive to undergo subsequent procedures are much more likely to survive their hospital stay.  However, predictors of death in ACS include increasing age, emergent presentation, and teaching hospital admission.  Consideration of more aggressive treatment of suspected ACS at an earlier stage for patients with these known high risk predictors may aide in saving lives.
 

11.05 Predicting Wound Complications by Pre-operative CT Measurements in Open Ventral Hernia Repair (OVHR)

Posted on December 22, 2014

S. W. Ross1, J. D. Watkins1, L. J. Blair1, A. E. Lincourt1, V. A. Augenstein1, B. T. Heniford1  1Carolinas Medical Center,Charlotte, NC, USA

Introduction: Pre-operative assessment of ventral incisional hernias is often aided by abdominal CT imaging.  We hypothesized that pre-operative CT measurements of defect and abdominal wall thickness(AWT) will correlate with complications and need for complex abdominal wall reconstruction(AWR).

Methods: A prospective, institutional, hernia specific database was queried for all OVHR from 2008-2012 and patients with preoperative CT of the abdomen/pelvis were selected. CT measurements were obtained of the hernia defect width, length, and AWT at multiple sites.  Standard statistical methods were used to evaluate the association of CT measurements with the need for component separation, panniculectomy, as well as wound complications, total complications and hernia recurrence.  Principal component analysis(PCA) was employed to analyze CT measurements and BMI.

Results: There were 151 OVHR with CT data.  In total, average: age 55.3±12.5 years, BMI 33.3±7.8 kg/m2, defect width 8.6±5.0cm, area 178.3±214cm2, AWT at umbilicus 3.5±1.8cm, and AWT at pubis 7.0±3.2.  The OVHR population was: 37.7% male, 60.3% were recurrent, 24.0% had a component separation and 34.4% required panniculectomy.  The rate of wound and total complications were 13.25% and 39.1% respectively.  Recurrence rate was 2.7%.  PCA was used to create a variable using six key CT measurements and BMI.  The analysis showed the following techniques or outcomes were associated with increasing CT measurements: component separation(defect width, length, and area); panniculectomy(all measurements excluding retrorenal); wound complications(all measurements excluding hip girdle and retrorenal); total complications(defect width, length, area and pubic symphysis AWT);all p<0.05.  Using multivariate regression, increasing PCA was independently associated with wound complications OR 1.08, CI (1.01-1.16).

Conclusion: Pre-operative CT measurements of hernia defects and abdominal wall thickness can predict wound complications and the need for complex AWR techniques.  Additionally, increasing measurements correlate significantly with hernia recurrence.  Consequently, PCA may be used in the future to calculate pre-operative risk and to predict the operative approach for OVHR.

 

1.01 PRINS, a long non-coding RNA is a marker of recurrence for adrenocortical cancer

Posted on December 22, 2014

A. R. Glover1, J. Zhao1, J. Ip1, J. Lee1, B. Robinson1, A. Gill1, P. Soon1, S. Sidhu1  1Kolling Institute Of Medical Research, Royal North Shore Hospital,University Of Sydney,Sydney, NSW, Australia

Introduction:

Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited treatment options and high rates of recurrence following surgical resection.  Long noncoding RNAs (lncRNAs) regulate multiple cell processes and have been implicated in the pathogenesis of adrenocortical carcinoma by exome sequencing and SNP analysis.  The aim of this study was to identify lncRNAs associated with ACC outcomes using dedicated lncRNA microarrays of adrenocortical tumours.

Methods:  

RNA was extracted from freshly frozen tissue with confirmation of diagnosis by histopathology.  Focused lncRNA transcriptome analysis was performed using the ArrayStar Human LncRNA V3.0 microarray to identify differentially expressed lncRNAs between ACC and normal adrenal cortex (NAC).  Selected lncRNAs were validated by qRT-PCR.  To identify possible lncRNAs associated with recurrence, median expression levels of differentially expressed lncRNAs were compared using the Mann-Whitney test between those with a clinical history of recurrence and non-recurrence.  From this analysis, lncRNAs were tested by qRT-PCR on an extended sample group to test this association with recurrence.  Area under the receiver operating characteristic curves (AUC) was used to determine accuracy for clinical outcomes.

Results: Microarray of 16 samples (10 ACC, 6 NAC) showed distinct patterns of lncRNA expression with 956 lncRNAs differentially expressed (Corrected P<0.05).  Of these 956 lncRNAs, 59 differentially expressed lncRNAs were found to be associated with ACC recurrence.  These 59 lncRNAs, included carcinogenesis implicated lncRNAs such as GAS5, FEZF1-AS1, RPL23AP82, GNAS-AS1, MALAT-1 and PRINS.  These carcinogenesis lncRNAs were tested by qRT-PCR on an extended  group (20 ACCs) where PRINS was confirmed to be differentially expressed between recurrence and non-recurrence groups (P<0.05).  Area under the receiver operating characteristic curve showed lower expression levels of PRINS was associated with recurrence (AUC=0.889, P=0.0044) and with metastatic disease on presentation (AUC=0.843, P=0.026).

Conclusions:

PRINS (psoriasis associated RNA induced by stress) has been implicated as a tumour suppressor in prostate cancer and may play a similar role in ACC. As expression levels of PRINS were found to be lower in samples associated with recurrence and the presence of metastatic disease at presentation it offers a novel prognostic and therapeutic target for further functional study.

1.02 Characterization of Anticancer Properties of a Novel HDAC Inhibitor in Carcinoid Cancer Cells

Posted on December 22, 2014

A. D. Harrison1, R. Jaskula-Sztul1, R. Nair1, A. Dammalapati1, G. Winston-McPherson2, C. M. Schienebeck2, K. Kupcho3, M. Robers3, W. Tang2, H. Chen1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Pharmacy,Madison, WI, USA 3Promega,Madison, WI, USA

Introduction: Carcinoid cancer consists of slowly growing neuroendocrine (NE) tumors within the gastrointestinal tract, which are often metastatic by the time of diagnosis making curative surgery infeasible.  They cause excessive production of various bioactive hormones resulting in the carcinoid syndrome and a poor quality of life for these patients.  The Notch pathway has proven to be tumor suppressive in NE cancer, resulting in a clinical need for therapeutic options to activate Notch.  Herein, we describe the novel and potent histone deacetylase (HDAC) inhibitor, AB3, which induces Notch signaling and alters the malignant neuroendocrine phenotype of carcinoid cancer.  We also fully characterized the activity and selectivity of AB3 against HDAC isoenzymes.

Methods: The cytotoxicity of AB3 on the GI carcinoid cell line (BON) was measured by determining the IC50 value via MTT assay (3-(4, 5-Dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide).  The effect of AB3 on cell death was investigated by two independent assessments: 1) PI stain-based sub-G1 cell fraction analysis and 2) detection of protein expression of apoptotic markers via Western blot (cyclin D1, survivin, XIAP and cleaved PARP).  Protein expression of NE tumor markers ASCL1 and CgA, and Notch1-3 isoforms were also detected by Western blot.  Luciferase assay for CBF1 binding (Notch pathway mediator) was used to measure the functional activity of Notch.  To characterize the activity and selectivity of AB3, HDAC-Glo TM assay and screening system was used.

Results: We demonstrated that the IC50 value for AB3 treatment in BON cells was 3 µM.  The mechanism of growth inhibition was found to be apoptosis, evident by an increased sub-G1 cell fraction (G0 DNA content).  In addition, protein expression of anti-apoptotic markers was decreased with concomitant cleavage of PARP.  Western blot analysis revealed ASCL1 and CgA reduction.  AB3 activated the Notch pathway as evident by an induction of Notch1- 3 isoforms.  Furthermore, CBF1 binding analysis revealed that the Notch pathway is activated.  We determined that AB3 exhibits selectivity for HDAC 1, 2, and 3, with less inhibitory potency against the HDAC 6, 8, and 10 isoforms.

Conclusion: We demonstrated that AB3 is a potent inhibitor of BON carcinoid cell proliferation at low µM concentrations.  The high antitumor activity of AB3 strongly suggests that this HDAC inhibitor may hold great therapeutic potential for the treatment of carcinoid cancer. 
 

1.03 Characterization of Histone Deacetylase Inhibitor Resistance in Metastatic Thyroid Cancer Cells

Posted on December 22, 2014

I. Lou1, X. Yu1, S. Jang1, A. Harrison1, H. Chen1  1University Of Wisconsin,Endocrine Surgery,Madison, WI, USA

Introduction:
Aberrant histone deacetylase (HDAC) activity has been shown to contribute to cancer development and HDAC inhibitors (HDACi) are currently under clinical investigation to treat a variety of malignancies.  Despite the promising drug efficacy shown in preclinical studies, most HDACi, including suberoylanilide hydroxamic acid (SAHA), have failed to demonstrate significant survival benefit for metastatic thyroid cancers in clinical trials. In our current study, we aim to delineate the possible mechanism responsible for HDACi resistance in metastatic thyroid cancer cells through the development and characterization of a SAHA-resistant cell line.

Methods:
FTC236 cells, a human follicular thyroid cancer derived cell line metastatic to the lymph nodes, were chronically exposed to SAHA at increasing concentrations and maintained in 2µM SAHA containing medium (FTC236/SAHA-R). Parental cells exposed only to dimethyl sulfoxide (DMSO), the solvent for SAHA, were used as control (FTC236/DMSO). Cell proliferation was then measured by viable cell count. HDACi resistance was evaluated by treating the two cell lines with different increasing concentrations of SAHA. Protein lysate was collected from FTC236/SAHA-R and FTC236/DMSO. Western blotting was performed to assess the expression of Notch 1 protein as well as epithelial mesenchymal transition (EMT) markers, which have previously been shown to play a role in cancer chemoresistance.

Results:
Based on the viable cell counts, the FTC236/SAHA-R cell line consistently showed a slower growth rate than FTC236/DMSO (221% vs. 550% at 72 hours, p<0.05). FTC236/SAHA-R was found to be resistant not only at the established drug concentration of 2 µM but also at higher concentrations. After 48 hours of SAHA exposure, 58% and 35% of FTC236/SAHA-R remained viable at 2 μM and 4 μM concentrations, respectively.  In comparison, only 35% and 23% of FTC236/DMSO survived in 2 μM and 4 μM SAHA, respectively. Analysis of the protein expression of these cell lines as well as the parental cell line revealed consistent silencing of Notch 1 signaling in the FTC236/SAHA-R cells. With respect to EMT markers, increased expression of Snail in FTC236/SAHA-R cells was observed compared with parental cell line and FTC236/DMSO while Slug remained unchanged.

Conclusion:
SAHA resistant thyroid cancer cells grow at a much slower rate. In addition, Notch 1 signaling is silenced and the expression of EMT marker Snail is increased in these resistant cells. This may shed light into possible mechanisms of HDAC inhibitor resistance and help with the development for new treatment targets.
 

1.04 The Novel PARP Inhibitor ABT-888 Enhances Dacarbazine Induced Cytotoxicity in Carcinoids in vitro

Posted on December 22, 2014

Y. R. Somnay1, S. Lubner2, H. Gill1, B. Matsumura1, H. Chen1  1University Of Wisconsin,Endocrine Surgery Research Laboratories, Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Human Oncology,Madison, WI, USA

Introduction:  Carcinoids are slow-growing neuroendocrine tumors that often present insidiously with few options for medical management. Monoagent DNA-alkylating chemotherapies such as Dacarbazine (DTIC) are among a paucity of therapies for these tumors, but are limited by host toxicity and intrinsic chemoresistance through the base excision repair (BER) pathway via the activity of poly (ADP-ribose) polymerase (PARP). Inhibitors of PARP are thought to potentiate DNA-damaging chemotherapies by blocking cancer cells’ ability to repair DNA double strand breaks following base excision repair. Here we show that the PARP inhibitor ABT-888 (veliparib) enhances the cytotoxic effects of DTIC in a pancreatic carcinoid cell line and potentiates DNA damage, cell cycle arrest and apoptosis. 

Methods:  Human pancreatic carcinoid cells (BON) were incubated in ABT-888 (0-20µM), DTIC (0-400µM) or both, for 96 hours. Cell growth was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. Western analysis showed expression levels of Chromogranin A (CgA) a well characterized marker for carcinoid malignancy, in addition to poly(ADP)-ribose polymerase (PARP), cleaved caspase 3, cyclin family kinases, and phosphorylated and total ataxia and telangiectasia mutated kinase (ATM), H2A histone family member X (H2AX), p53 and retinoblastoma gene (Rb). Flow cytometry using propidium iodide staining was used to assess cell cycle kinetics.  

Results: Cell survival after treatment with ABT-888 and DTIC was reduced synergistically as combination indices (CI) fell below 1 on the Chou-Talalay scale, as we've previously reported. Notably, ABT-888 alone was non-toxic at therapeutic doses. ABT-888 administered prior to varying DTIC doses reduced CgA expression beyond the combined effect of either drug alone. Of note, the degree of ATM and H2AX phosphorylation, indicative of double strand DNA breaks, was enhanced by ABT-888 prior to DTIC treatment, suggesting BER pathway attenuation via PARP inhibition. This was corroborated by an increase in phospho-p53, a tumor suppressor activated by DNA damage, alongside reductions in cyclin D and downstream inactive phospho-Rb. ABT-888 treatment prior to DTIC decreased levels of cyclin A expression, and accordingly augmented G2 and G0-G1 phase arrest, indicating an enhanced transition into apoptosis. Finally, ABT-888 potentiated DTIC-induced cleavage of terminal apoptotic markers caspase 3 and PARP relative to the effect exerted by either monotherapy.  

Conclusion: ABT-888 synergizes with DTIC to suppress carcinoid growth and phenotype. By targeting PARP activity, ABT-888 may impair BER pathway response to DTIC, inducing a futile cycle of persistent DNA strand breakage and potentiating cell cycle arrest and apoptosis through p53 by enhancing Rb hypophosphorylation. By targeting DNA repair pathways that confer cancer cell resistance, ABT-888 may help treat carcinoids that remain refractory to mainstay therapies.

1.05 MicroRNA-21 Regulates Melanoma Invasion via Inhibition of Tissue Inhibitor of Metalloproteinases-3

Posted on December 22, 2014

N. Latchana3, S. Martin Del Campo3, V. Grignol3, K. Levine3, E. Fairchild6, A. Ganju3, C. Jaime-Ramirez3, T. Dao5, V. Karpa5, M. Carson3, A. Chan5, W. Carson3  3Ohio State University,Columbus, OH, USA 5Wright State University,Dayton, OH, USA 6Nationwide Children’s Hospital,Columbus, OH, USA

Introduction: Melanoma accounts for the highest number of skin cancer associated deaths annually yet the progression towards a metastatic phenotype is not completely understood.  Increased cellular invasion through the extracellular matrix is a prerequisite for metastasis and is enhanced by matrix metalloproteinases (MMPs), MMPs are negatively regulated by the tissue inhibitor of metalloproteinases (TIMP) protein family such as TIMP3.  MicroRNAs (miRs) are small, noncoding RNAs that inhibit gene expression and regulate many cellular processes.  It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma.  It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby promote the invasiveness of melanoma cells.

Methods: WM1552c, WM793b, A375 and MEL39 melanoma cell lines were transfected with control miR, pre-miR-21, or anti-TIMP3.  Oligonucleotide uptake was assessed by Real-Time PCR and transfection efficiency was determined using fluorescent microscopy with a FAM-miR construct during transfection.  Transfected cells were used for invasion, proliferation, and migration assays.  Immunoblot analysis was performed on transfected cells for targets of miR-21 including programmed cell death protein (PDCD4), tropomyosin-1 (TM1), phosphatase and tensin homolog (PTEN).  A375 cells were transfected with oligonucleotides against miR-21 or a control miR before injection into the flanks of 01B74 Athymic NCr-nu/nu mice.  Tumor growth was analyzed over three weeks.  Another group of mice were injected with untransfected A375 cells in the flank and monitored until tumor growth reached an average of 100mm3 before injections with PBS alone (control) or anti-miR 21 every three days for four cycles.  Tumor growth was monitored and tumor specimens were analyzed for TIMP3 expression by immunohistochemistry using a goat anti-TIMP3.

Results: Fluoroscopic evaluation revealed >90% transfection efficiency of miRs into melanoma cells with oligonucleotide uptake also confirmed by PCR.  Immunoblot analysis of miR-21 overexpressing cells revealed reduced expression of TIMP3.  This in turn led to an increase in the invasiveness of the 4 melanoma cell lines as wells as an additional melanoma cell line, 1174 MEL.  miR-21 transfection did not change the proliferation or migration capacity of melanoma cells.  Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cells, mimicking the effects of miR-21 over-expression.  Treatment of tumor cells in vivo with an antagomir to miR-21 inhibited tumor growth with a corresponding increased expression of TIMP3 on immunohistochemistry.  Intra-tumoral injections of anti-miR-21 in vivo produced similar effects.

Conclusion: Increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.

 

1.06 Suppression of CXCL10/CXCR3 Switches Polymetastatic Phenotype to Oligo- in a Melanoma Mouse Model

Posted on December 22, 2014

S. C. Wightman1, A. Uppal1, G. Oshima1, X. Huang1, S. Ganai2, N. N. Khodarev1, M. C. Posner1, R. R. Weichselbaum1  1University Of Chicago,Chicago, ILLINOIS, USA 2Southern Illinois University,Carbondale, ILLINOIS, USA

Introduction:   Oligometastasis is a state of limited metastasis with the potential for long-term disease free control by surgery or radiotherapy. We designed syngeneic mouse melanoma models with oligo-  and polymetastatic pulmonary disease. We found CXCL10, an interferon inducible chemokine that acts on the receptor CXCR3, consistently elevated in polymetastatic tumor clones both in vivo and in vitro.

Methods:   Two stable CXCL10 KDs (knockdowns) and two stable CXCR3 KDs of both oligo- and polymetastatic B16F1 clones (P2M5B and P2M3C respectively) were generated by lentiviral transfection and suppression confirmed by Western blot.  Lung metastases were counted 2.5-3.5 weeks after tail-vein injection of the KD clones and non-targeting controls (NTCs). Flank primary tumors were generated via injection of the KD clones or NTCs and measured serially over 19 days. 

Results:  The P2M3C CXCL10 KD #1 and KD #2 had decreased metastases, 27.7+/-21.1 and 26.7+/-16.9, compared to P2M3C-NTC with 89.7+/-14.3 metastases (p<0.001).  Similarly, the P2M5B CXCL10 KD #1 and KD #2 had decreased metastases, 9.4+/-6.2 and 2.8+/-1.6, compared to P2M5B-NTC with 25.4+/-11.0 metastases (p<0.01).  For CXCR3, the P2M3C CXCR3 KD #1 and KD #2 had decreased metastases, 86.1+/-32.3 and 77.3+/-19.8, compared to P2M3C-NTC with 134.4+/-39 metastases (p = 0.03 and 0.004 respectively).  The CXCR3 P2M5B KD #2 had decreased metastases to 1.7+/-0.8, compared to P2M5B-NTC with 31.3+/-25.5 metastases (p=0.02).  The CXCR3 P2M5B KD #1, while with a decreased number of metastases, was not statistically significant with 14.7+/-7.5 (p = 0.12). After experiments were done with KDs, primary tumors were grown in the flanks of mice to identify differences in the CXCL10 KDs outside of tumor metastases.  At day 19, volume in both of the P2M3C CXCL10 KDs were decreased with KD #1 having an average volume of 0.31+/-0.29 cm3 and KD #2 having an average volume of 0.15+/-0.15 cm3 compared to the P2M3C-NTC with a volume of 4.94+/-1.63 cm3 (p<0.001 for both).  The P2M5B CXCL10 KDs also both decreased with KD #1 having an average volume of 2.10+/-0.73 cm3 and KD #2 0.60+/-0.07 cm3 compared to the P2M5B-NTC with a volume of 7.81+/-1.80 cm3 (p = 0.03 and 0.001 respectively).

Conclusion:  Our experiments stress the vital role of the CXCL10/CXCR3 axis in metastases generation and primary tumor growth in our murine melanoma model.  As noted above, knocking down the CXCL10/CXCR3 axis is able to change the phenotype from polymetastatic to oligometastatic effecting the ability for metastatic tumor colonization. In primary tumors, decreasing the CXCL10 levels decrease tumor growth.  This potentially offers a gateway for therapeutic intervention in patients with melanoma.  These findings stress the importance of CXCL10 in tumor biology for both metastases and primary tumors in murine melanoma.  Further experiments are needed to dissect mechanisms through which CXCL10 regulates pulmonary metastases.

 

1.07 CDK4/6 inhibitor, PD-0332991 Synchronizes Sarcoma Cells for Synergistic Demise with WEE1 Inhibitor, MK-1775

Posted on December 22, 2014

A. M. Francis1, A. Alexander2, J. P. Carey2, V. Ravi3, K. Keyomarsi2, K. K. Hunt1  1University Of Texas MD Anderson Cancer Center,Surgical Oncology,Houston, TX, USA 2University Of Texas MD Anderson Cancer Center,Experimental Radiation Oncology,Houston, TX, USA 3University Of Texas MD Anderson Cancer Center,Sarcoma Medical Oncology,Houston, TX, USA

Introduction:
The retinoblastoma (RB) gene pathway is one of the most frequently altered pathways in human cancer. This pathway is regarded as one of the central regulators of cell proliferation through the coordination of the RB protein, cyclin-dependent protein kinases (CDK4, CDK6), D-type cyclins, the INK4 family of cyclin-dependent kinase inhibitors and the E2F-family of transcription factors. CDK4/6 inhibitors such as PD-03329991 (PD-991) have been shown to cause a G1 arrest in RB-positive cells while WEE1 inhibitors such as MK-1775 can cause premature mitotic entry of S-phase cells. We hypothesized that sarcoma cells could be synchronized in S-phase using PD-991 followed by a period of recovery to facilitate synergistic demise with MK-1775.

Methods:
Sarcoma cell lines (HT-1080, SK-LMS-1, SaOS-2) were evaluated for RB-pathway alterations using western blot analysis. Cells were treated with PD-991 for 6 days and MK-1775 for 2 days to determine IC50 values. FACS analysis was used to determine the optimal recovery timepoint for synchronization in S-phase after treatment with PD-991. Combination experiments involved application of PD-991 with or without a period of recovery followed by application of MK-1775. CalcuSyn 2.0 was used to yield a combination index (CI) to determine if the combination was synergistic (CI<1), additive (CI=1) or antagonistic (CI>1).

Results:
HT-1080 and SK-LMS-1 were RB-positive while SaOS-2 was RB-negative. PD-991 was more effective as single agent therapy in RB-positive cells (1.5-2.4uM) than RB-negative cells (7.0uM). MK-1775 had similar efficacy across all cell lines tested (1.4uM [HT-1080], 0.7uM [SK-LMS-1], 1.0uM [SaOS-2]). The greatest proportion of S-phase cells was observed after 6-9 hours of recovery from PD-991 (HT-1080: 21-25% vs 16% [vehicle], SK-LMS-1: 30-43% vs 21% [vehicle], SaOS-2: not obtainable due to its RB-negativity). PD-991 followed by MK-1775 was strongly synergistic in RB-positive cells (Figure). A period of recovery after treatment with PD-991 enhances this synergism. In RB-negative SaOS-2, the combination of PD-991 and MK-1775 was additive to antagonistic (CI 1.0-1.5).

Conclusion:
Synchronization of cells in S-phase using PD-991 followed by treatment with MK-1775 causes synergistic demise of RB-positive sarcoma cells. In vivo studies are warranted to test this novel treatment strategy and are currently underway.
 

1.08 GPR40 as a therapeutic target in melanoma and neural crest-derived tumors

Posted on December 22, 2014

M. I. Chang1,2, P. Nandivada1,2, S. J. Carlson1,2, A. Pan1,2, M. Puder1,2  1Boston Children’s Hospital,Surgery,Boston, MA, USA 2Boston Children’s Hospital,Vascular Biology Program,Boston, MA, USA

Introduction: Melanoma is a neural crest derived tumor and is the most deadly form of skin 
cancer. In advanced stages, this and other neural crest tumors (e.g., medullary thyroid, 
malignant peripheral nerve sheath tumor, neuroblastoma, etc) have limited treatment options as 
they often manifest as aggressive disease. Recent studies have demonstrated cellular 
apoptosis in these tumors in the presence of the omega-3 fatty acid, docosahexaenoic acid 
(DHA). The purpose of this study is to determine if 1) this effect is mediated through the free 
fatty acid G-protein coupled receptor 40 (GPR40) and 2) determine if selective agonism of 
GPR40 produces cell death in vitro. 

Methods: The presence of the GPR40 receptor was confirmed by PCR and Western blot 
analysis on neural crest tumor cell lines including: neuroblastoma, melanoma, medullary thyroid 
carcinoma, and malignant peripheral nerve sheath tumor. These cell lines were treated with 
varying concentrations of DHA and TAK-875 (a specific, high affinity GPR40 agonist) for 3 and 6 
days while using a human fibroblast cell line as a control. Cell viability was determined using an 
absorbance-based cell viability assay. 

Results: DHA exhibited a minimal inhibitory affect against medullary thyroid carcinoma. 
However, cell death was observed in neuroblastoma, melanoma, malignant peripheral nerve 
sheath tumor, and fibroblasts at high DHA concentration. TAK-875 produced a profound 
selective inhibitory effect on cell proliferation and promoted cell death of all of the neural crest 
derived tumor cell lines without toxicity in the control fibroblast line at nanomolar concentrations. 
Interestingly, no GPR40 expression was noted in the malignant peripheral nerve sheath tumor. 

Conclusion: GPR40 agonist TAK875 produces profound selective cell growth inhibition and 
death in neural crest derived tumor cell lines, with reduced cellular toxicity. While the GPR40 
receptor may serve as a novel target in the treatment of neural crest-derived tumors, the affect 
of this drug on the malignant peripheral nerve sheath tumor may indicate that GPR40 alone may 
not be responsible for this dramatic response in cell viability in neural crest-derived tumor cell 
lines. 

1.09 Notch2 Has an Opposing Role to Other Notch Isoforms in Neuroendocrine Tumors

Posted on December 22, 2014

T. V. Do1, A. Dammalapati1, A. Hundal1, H. Jin1, R. Jaskula-Sztul1, H. Chen1  1Department Of Surgery,Madison, WI, USA

Introduction: Notch signaling involves in various aspects of mammalian biology such as cellular differentiation, cell-cycle regulation, metabolism and apoptosis. Among the four identified mammalian Notch receptors, the role of Notch1 and 3 as tumor suppressor in neuroendocrine tumor cells (NET) have been elucidated. Nevertheless, the function of Notch2 signaling still remains unclear in NETs. The aim of this study was to access the role of Notch2 in gastrointestinal (GI)NETs (carcinoids).

Methods: pcDNA4/V5-His plasmid ,containing constitutively expressed human active portion of Notch2 (NICD2), was transiently transfected into GI carcinoid (BON) cells. The same plasmid without NICD2 was used as a control. Transfection efficiency was assessed by co- transfection with plasmid expressing the green fluorescent protein (GFP). The expression of NICD2 was confirmed by quantitative RT-PCR and Western Blot. Next the functional activity of NICD2 was analyzed by measuring the degree of CBF-1 binding by luciferase reporter assay. Cell viability was then tested by MTT (3-(4, 5-Dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide) assay after 24, 48, 72 and 96 hours after transfection. To investigate the potential effects of NICD2 on BON cells proliferation, cell cycle and anti-apoptotic markers such as X-linked inhibitor of apoptosis (XIAP), cyclin D1, and c-Myc were examined by Western Blot analysis.

Results: The expression of NICD2 was detected over all time points of transfection on both mRNA and protein levels. CBF-1 binding assay showed increased luciferase activity indicating functional activation of NICD2. In contract to Notch1 and 3 which are tumor suppressive, NICD2 transfected cells did not reduce proliferation comparing to the cells transfected with control plasmid. Moreover, Western blot analysis showed an increase of anti- apoptotic markers XIAP, C-Myc, and Cyclin D1 with the overexpression of NICD2.

Conclusion: Carcinoid tumor cells have paucity Notch2.  For the first time, we demonstrate the growth – promoting function of Notch2 receptor in carcinoid cancer with concomitant upturn of anti-apoptotic markers XIAP, c-Myc and cyclin D1.  This opposing role of Notch2 isoform in NE cancer progression warrant further investigation.

 

1.10 Cdk5/p25 Regulates Notch1 and Notch2 Intracellular Domains in Medullary Thyroid Carcinoma

Posted on December 22, 2014

S. K. Odorico1, X. Yu1, A. Dammalapati1, A. Harrison1, A. Hundal1, J. A. Bibb2, H. Chen1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Texas Southwestern Medical Center,Department Of Psychiatry,Dallas, TX, USA

Introduction:  Medullary thyroid cancer (MTC) is a neuroendocrine carcinoma that arises from C cells.  Recently, it has been demonstrated that Cdk5 and its cofactor p25 promote C cell proliferation, leading to the development of MTC, while repressing p25 overexpression causes C cell growth arrest.  In our previous studies, we have shown that activation of Notch intracellular domain inhibits MTC cell proliferation and alters the neuroendocrine phenotype.  However, little is known about the possible interaction between Cdk5/p25 and pan Notch signaling.  Therefore, the purpose of this study was to investigate the role of Notch isoforms including Notch1 and 2 in the p25–mediated inhibition of proliferation in MTC cells.

Methods:  A murine thyroid cancer cell line MTCp25, which retains TetOp promoter-controlled p25-GFP expression, was used in our study.  The exogenous p25-GFP is stably overexpressed under basal conditions and repressed by treatment with doxycycline.  We treated MTCp25 cells with 3 different concentrations of doxycycline (1, 2 and 5 μg/mL) for 2, 4 and 6 days.  Notch1, Notch 2, p25, GFP and beta actin expression were then evaluated by Western blot.  We also quantified the mRNA levels of Notch isoforms during different time points using quantitative real-time polymerase chain reaction (PCR).

Results: Both p25 and GFP expression were consistently reduced during different time points in MTCp25 cells culturing in the presence of doxycycline.  No significant changes were detected by Western blot for the intracellular domain of Notch 1 or Notch2 after 2-day treatment of doxycycline.  Interestingly, protein expression of Notch1 and Notch2 intracellular domains profoundly increased in a dose dependent manner after repressing p25 for 4 and 6 days.  Furthermore, we found that the mRNA levels of Notch 1 and Notch 2 did not change significantly with p25 suppression, suggesting that p25 may be involved in the regulation of protein cleavage or degradation of Notch1 and Notch2.

Conclusion: Cdk5/p25 signaling up-regulates the protein level of Notch1 and Notch2 intracellular domains.  This intriguing finding could suggest the function of p25 involved in the post-translational processing of Notch1 and Notch2 receptor.  These results call for further investigation into the mechanism on the crosstalk between Notch and Cdk5 signaling pathways, which may play an important role in medullary thyroid cancer development and growth.

 

1.11 Association of Rosacea and Angiosarcoma/Lymphangiosarcoma (AS/LAS)

Posted on December 22, 2014

S. P. Olsen1, M. C. Perez2, A. M. Priddy1, E. S. Armbrecht1, A. K. Behera1, S. W. Fosko1, N. G. Zeitouni3, D. E. Winstead4, F. E. Johnson1  1Saint Louis University School Of Medicine,St. Louis, MO, USA 2Moffitt Cancer Center And Research Institute,Surgery,Tampa, FL, USA 3Roswell Park Cancer Institute,Dermatology,Buffalo, NY, USA 4Sarcoma Foundation Of America,Burlington, NC, USA

Introduction: Rosacea-affected tissue has increased angiogenesis/lymphangiogenesis.  Isolated case reports have intimated that rosacea, and particularly rhinophyma (a late manifestation of rosacea), might cause angiosarcoma (AS) or lymphangiosarcoma (LAS) in rosacea-affected tissue.  As in Stewart-Treves syndrome, AS/LAS feature prominent varicose veins, lymphatic obstruction, edema, and other features of chronic inflammation.  No reports aimed at quantitatively establishing a causal relationship have been published, as far as we are aware.  Recent evidence indicates that an aberrant innate immune response, mediated by altered endogenous polypeptides known as cathelicidins, is a primary pathologic event in rosacea.  They are pro-inflammatory and vasoactive agents that cause the excessive inflammation in rosacea.  Thus it is plausible that rosacea may be a risk factor for AS/LAS.  We carried out an observational study to confirm or refute this possibility.

Methods: IRB approval was obtained.  We developed a simple one-page data sheet to abstract data from pathology reports of patients with head and neck sarcomas of all sub-types.  We excluded Kaposi sarcoma, Ewing sarcoma, carcinoma, and sarcoma not in the head and neck region.  There were entries for other possible causes of sarcoma (prior radiation exposure, genetic predisposition, etc.).  We obtained data from several large cancer centers and calculated the crude odds ratio in this classic retrospective case-control study.

Results: Data were analyzed in “waves” corresponding to the data sources.  Wave 1 had 228 cases from 3 centers, of which 198 met inclusion criteria.  The odds ratio for the association between rosacea and AS/LAS was 11.9 (95% CI: 1.05, 136.0).  This is statistically significant.  The confidence interval is wide.  Wave 2 included 53 cases from one cancer center, of which all met the inclusion criteria.  The odds ratio for the association between rosacea and AS/LAS was 0.56 (95% CI: 0.08, 3.72).  This is not statistically significant.  Combining wave 1 and wave 2, we had 251 evaluable cases.  The odds ratio for the association between rosacea and AS/LAS was 4.76 (95% CI:  1.11, 20.50).  This is statistically significant but the confidence interval is wide.

Conclusion: This is the first quantitative analysis designed to measure the association between rosacea and head and neck angiosarcoma/lymphangiosarcoma.  A statistically significant association was observed but the confidence interval surrounding the odds ratio is wide.  We seek access to other large data sets for additional analyses.

1.13 Exosomal miRNA Mediated Intercellular Communication of Thyroid Cells

Posted on December 22, 2014

J. C. Lee1,2, J. T. Zhao1, L. Bach2, J. Ip1, J. Gundara1, A. Glover1, J. Serpell3, S. Sidhu1  1University Of Sydney,Kolling Institute Of Medical Research,Sydney, NSW, Australia 2Monash University,Melbourne, VIC, Australia 3The Alfred Hospital,Department Of Surgery,Melbourne, VIC, Australia

Introduction: The potential for circulating miR-146b and miR-222 in papillary thyroid cancer (PTC) patients to be used as circulating biomarkers has recently been reported. This study aimed to demonstrate the presence of miR-146b and miR-222 in PTC-derived exosomes, and to investigate the potential role of these exosomes in an in vitro model. 

Methods: Exosomes were isolated from the condition medium of PTC cells. Western blot of CD9 surface marker was used to confirm the isolation of exosomes. The miRNA contents of PTC cells, conditioned medium and exosomes were compared. Benign thyroid and PTC cells were treated with PTC-derived exosomes, and the changes in their proliferation were measured by the MTT assay.

Results:The isolated exosomes were positive for the CD9 surface marker on Western blots. The concentration of miR-222 was similar in the exosomes and PTC cells, but lower in the conditioned medium. On the other hand, the concentration of miR-146b was highest in the cells and lowest in the conditioned medium, with concentrations within the exosomes somewhere in between. Treatment with PTC-derived exosomes resulted in reduced proliferation of both benign and malignant thyroid follicular cells.

Conclusion:The results demonstrated that miR-146b and miR-222 are both secreted by PTC cells, packaged in exosomes. These can be used as circulating biomarkers. It appears that PTC cells may release exosomes that mediate a reduction of proliferation in other cells. This in turn would confer a survival advantage for the PTC cells. 
 

1.14 Targeting Sirtuins Blocks Neuroblastoma Cell Proliferation And Induces Differentiation

Posted on December 22, 2014

E. J. Rellinger1, H. Song2, S. Park2, P. Paul1, B. T. Craig1, J. Qiao1, V. Athanasios2, D. R. Gius2, D. H. Chung1  1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA 2Northwestern University,Radiation Oncology,Chicago, IL, USA

Introduction:  Neuroblastoma (NB) is a pediatric solid tumor that arises from failed differentiation of neural crest progenitors. Infants and children with high-risk NB have a poor prognosis despite our most aggressive therapies, highlighting the need for novel drug targets. Sirtuins are a family of seven NAD+-dependent histone deacetylases that have emerged as key regulators of cell fate, DNA damage repair, neuronal protection, and tumorigenesis. However, the role of Sirtuins in NB tumorigenicity is unknown. The purpose of this study was to evaluate the exact role of Sirtuins in regulating NB cell proliferation and differentiation. 

Methods:  Sirtuin inhibition in BE(2)-C human NB cells was performed using nicotinamide (NAM), a nonspecific Sirtuin inhibitor, or shRNA transfection (shSIRT1, shSIRT2, shSIRT3, shSIRT6, shSIRT7). Cell viability was determined using CCK-8 assays. Confocal microscopy and immunoblotting with neuron specific enolase (NSE) and neurofilament-M (NF-M) were employed to assess for neural differentiation. Rescue experiments in shSirt6 cells were completed with pCDH-SIRT6 transfection. Sirt6 expression in human tumor sections was assessed using immunohistochemistry and counterstained with H&E to score histologic grade. Cell cycle analyses were completed using flow cytometry. Anchorage-independent soft agar colony growth was used for in vitro representation of tumor invasiveness.

Results: NAM treatment decreased cell viability, induced morphologic differentiation, and increased protein expression of p21 (CDK inhibitor) and NSE, our respective markers of cell cycle arrest and neural differentiation. Only transfection with shSirt6 similarly decreased proliferation and induced cell morphology and neural marker expression patterns consistent with neural differentiation in BE(2)-C cells (Fig. A). Silencing Sirt6 induced NB cell arrest at G0/G1 cell cycle phase and inhibited DNA synthesis and soft agar colony formation. Rescue of cell proliferation, morphologic differentiation, cell cycle arrest, and protein expression was achieved using pCDH-SIRT6 transfection (Fig. B). Sirt6 expression was decreased in differentiated human NB sections. Retinoic acid treatment diminished Sirt6 expression and induced differentiation synergistically with Sirt6 silencing. 

Conclusion: Targeted inhibition of Sirt6 in NB downregulates tumor growth and promotes neural differentiation synergistically with retinoic acid treatment. These findings suggest that Sirt6 is a novel therapeutic target in NB and highlights clinical strategies to optimize its efficacy.

 

1.15 Establishment of an Orthotopic Xenograft Model of Metastatic Neuroblastoma Using Ultrasound Guidance

Posted on December 22, 2014

R. Van Noord1, D. Bashllari1, M. Hoenerhoff2, E. A. Newman1  1University Of Michigan,Pediatric Surgery,Ann Arbor, MI, USA 2University Of Michigan,In Vivo Animal Core, Pathology Core,Ann Arbor, MI, USA

Introduction:  Neuroblastoma (NB), the most common cancer in infancy arises from neural crest precursors in the adrenal medulla and sympathetic neural ganglia. The majority of tumors arise in the adrenal gland. Nearly 50% of patients have high-risk Stage IV disease that relapses after standard multimodality therapy.  In order to better understand NB tumor biology and to examine novel therapeutic approaches, cancer researchers often utilize animal xenograft models.  Surgical models are often tedious and labor intense with pain and long recovery periods.  Further limitations of previous models include slow rate of tumor formation, inappropriate microenvironment, and lack of metastases. We hypothesized that a minimally invasive technique utilizing high-resolution ultrasound guided needle injections of tumor is a safe and efficient method to establish orthotopic xenograft models of NB for preclinical studies. 

Methods: Real-time ultrasound procedures were performed utilizing the VisualSonics’ Vevo 770 in vivo imaging system. Six week-old immune deficient mice (NOD-SCID) were anesthetized in an induction chamber. A chilled Hamilton syringe fitted with a 27 G needle loaded with luciferase-tagged human NB cells (SH-SY5Y or IMR-32) suspended in matrigel was guided into the adrenal gland capsule utilizing ultrasound guidance.  Cell suspensions were hand injected into the adrenal gland, above the kidney and below the liver or spleen. Tumor growth was measured weekly via bioluminescence.  Tumors were analyzed by veterinary science pathologists in the Pathology Cores for Animal Research, and characterized using paraffin-embedded formalin-fixed slides stained for H/E, neuron specific enolase, and tyrosine hydroxylase. A total of 18 animals were studied (SH-SY5Y and IMR-32).  All procedures were followed and approved by the University Committee on Use and Care of Animals.

Results: There was minimal animal recovery post-procedure, and no morbidity or mortality. In all animals, NB tumors successfully engrafted at the suprarenal space confirmed by weekly imaging via bioluminescence and histological characterization.  There was evidence of local tumor engraftment by seven days post-injection.  Tumors progressed to bulky local disease with distant metastases by four weeks.  Metastatic sites included lymph nodes and bone, sites typical of NB spread.

Conclusions: We demonstrate that a novel metastatic murine model of NB is established by a percutaneous, ultrasound guided technique. Tumors are detected, monitored and quantified prior to visualization using bioluminescence.  This minimally invasive technique is target specific, safe, and efficient, making it an optimal metastatic preclinical model to test new therapeutic agents for high-risk neuroblastoma.

1.16 Valinomycin Has Anti-tumor Activity Against Neuroblastoma Cell Lines Through Down-regulation of MYCN

Posted on December 22, 2014

R. B. Interiano1,2, J. Yang1, D. Hu1, N. Hinkle1,2, C. Morton1, A. M. Davidoff1,2  1St. Jude Children’s Research Hospital,Surgery,Memphis, TN, USA 2University Of Tennessee Health Science Center,Surgery,Memphis, TN, USA

Introduction: Neuroblastoma is the most common solid, extra-cranial, pediatric malignancy, and MYCN amplification has been associated with poor prognosis in this tumor type.  Valinomycin is a potassium ionophore which has been shown to cause significant deregulation of intracellular potassium.  In a preliminary comparative drug study, we noted valinomycin to have potential effect on tumors with down-regulated MYCN.  We sought to assess the antitumor effect of this drug on the expression of MYCN.

Methods: MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines SK-N-BE2, IMR32, NB1691, SK-N-AS, and SK-N-SH were treated with valinomycin with a range of doses (0, 0.001, 0.01, 0.1, 1, 10 μM) for variable time courses.  Cell viability was assessed by Presto Blue assay and protein expression of MYCN was assessed via western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR).  Time courses of treatment with valinomycin at 10nM were then used to assess apoptosis with western blot.  Mice with flank tumors were then treated at the maximum tolerated dose of 0.5 mg/kg, and were assessed for therapeutic effect

Results:Valinomycin had a significant antitumor effect with >50% cell death in all neuroblastoma cell lines following treatment at 10nM for 72 hours.  There was a significant decrease in protein expression of MYCN and c-myc in non-MYCN-amplified lines, except SK-N-AS, occurring as early as 2-4 hours, prior to PARP cleavage and apoptosis occurring. There was no significant change in MYCN mRNA at 24-hour treatment with valinomycin at 10nM.  Valinomycin given to xenograft bearing MYCN-amplified tumors showed slowing of progression in SJ-NB12 (~30% at 3 weeks); however, no therapeutic effect was noted in SK-N-AS or NB1691.

Conclusion: Valinomycin has a significant antitumor effect on neuroblastoma cell lines in vitro.  This antitumor effect is in part through the down-regulation of MYCN, prior to apoptotic signaling.  Understanding the mechanism of targeting MYCN down-regulation may lead to improved therapeutics.  Drug toxicity likely does not allow an appreciable pharmacodynamic effect on in vivo neuroblastoma tumors, although preliminary results suggest it may cause growth delay in some tumors.

 

1.17 A Role For MicroRNA-21 And Long Non-Coding RNA GAS5 In MYCN Amplified Neuroblastoma

Posted on December 22, 2014

C. Courtney2, J. Hu3, J. Xu3, K. Liechty3, R. C. Burns2, T. Westmoreland2  2Nemours Children’s Hospital; University Of Central FL,General Surgery,Orlando, FL, USA 3Children’s Hospital Colorado,General Surgery,Aurora, CO, USA

Introduction: MYCN amplification portends a poor prognosis in neuroblastoma patients and is associated with increased tumor growth, but MYCN amplification does not always equate to increased MYCN gene expression.  Regulation of growth occurs at many levels, including proliferation and the genes to regulate this process.  Recent novel regulators of proliferation have been identified and include long noncoding RNAs (lncRNA) and micro RNAs (miRNA).  Specifically, the lncRNA growth arrest-specific 5 (GAS5) and its negative regulator miRNA-21 have been implicated in the regulation of proliferation during tumor growth.  We hypothesized that lncRNA GAS5 and miRNA-21 would be differentially expressed in MYCN amplified and MYCN non-amplified neuroblastoma cell lines.

Methods: To test this hypothesis, total cellular RNA was isolated from the human neuroblastoma cell lines SK-N-AS (MYCN non-amplified) and IMR-32 (MYCN amplified).  The expression of the lncRNA GAS5 and miRNA 21 were measured using real-time reverse transcriptase polymerase chain reaction (QPCR).  The proliferation rates of both cell lines were also quantified using the MTT cell proliferation assay and compared to gene expression.  In addition, miRNA 21 was ectopically expressed in a lentivirus and knocked down utilizing an antagomiR.  Expression of miRNA 21 and lncRNA GAS5 was measured using QPCR.

Results: The expression of lncRNA GAS5 is increased in the IMR-32 (MYCN amplified) as compared to the SK-N-AS (MYCN non-amplified).  miRNA 21 expression demonstrates an inverse relationship to lncRNA GAS 5 in these cell lines.   In the IMR-32 cell line, ectopic expression of miRNA 21 resulted in a decreased expression of lncRNA GAS5.  The inverse was also true.  When the antagomiR for miRNA 21 was transfected into the IMR-32 cell line, the lncRNA GAS5 expression increased.  Proliferation rates were decreased in the IMR-32 cell line as compared to the SK-N-AS cell line.

Conclusion: miRNA 21 and lncRNA GAS5 are differentially expressed in neuroblastoma cell lines with and without MYCN amplification.  The inverse correlation of miRNA 21 and lncRNA GAS5 demonstrated in our neuroblastoma study has also been described in other tumors.  It is known that accumulation of lncRNA GAS5 results in decreased levels of apoptosis-related genes, cIAP2 and SGK1.  The inactivation of these genes results in reduced apoptosis.  Furthermore, reduced proliferation, as shown in our study, has been associated with reduced apoptosis. This work gives mechanistic insight to the regulation of apoptosis in MYCN amplified neuroblastoma.

 

« Previous Page
Next Page »